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Yorodumi- EMDB-6334: Negative stain 3D reconstruction of the yeast 26S proteasome in A... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-6334 | |||||||||
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Title | Negative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein | |||||||||
Map data | Negative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein | |||||||||
Sample |
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Keywords | Proteasome / UPS / Ubp6 / deubiquitinase / regulatory particle | |||||||||
Function / homology | proteasome regulatory particle / Proteasome, subunit alpha/beta Function and homology information | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 25.2 Å | |||||||||
Authors | Bashore C / Dambacher CM / Matyskiela M / Lander GC / Martin A | |||||||||
Citation | Journal: Nat Struct Mol Biol / Year: 2015 Title: Ubp6 deubiquitinase controls conformational dynamics and substrate degradation of the 26S proteasome. Authors: Charlene Bashore / Corey M Dambacher / Ellen A Goodall / Mary E Matyskiela / Gabriel C Lander / Andreas Martin / Abstract: Substrates are targeted for proteasomal degradation through the attachment of ubiquitin chains that need to be removed by proteasomal deubiquitinases before substrate processing. In budding yeast, ...Substrates are targeted for proteasomal degradation through the attachment of ubiquitin chains that need to be removed by proteasomal deubiquitinases before substrate processing. In budding yeast, the deubiquitinase Ubp6 trims ubiquitin chains and affects substrate processing by the proteasome, but the underlying mechanisms and the location of Ubp6 within the holoenzyme have been elusive. Here we show that Ubp6 activity strongly responds to interactions with the base ATPase and the conformational state of the proteasome. Electron microscopy analyses reveal that ubiquitin-bound Ubp6 contacts the N ring and AAA+ ring of the ATPase hexamer and is in proximity to the deubiquitinase Rpn11. Ubiquitin-bound Ubp6 inhibits substrate deubiquitination by Rpn11, stabilizes the substrate-engaged conformation of the proteasome and allosterically interferes with the engagement of a subsequent substrate. Ubp6 may thus act as a ubiquitin-dependent 'timer' to coordinate individual processing steps at the proteasome and modulate substrate degradation. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_6334.map.gz | 40 MB | EMDB map data format | |
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Header (meta data) | emd-6334-v30.xml emd-6334.xml | 12 KB 12 KB | Display Display | EMDB header |
Images | 400_6334.gif 80_6334.gif | 31.6 KB 3.8 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-6334 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-6334 | HTTPS FTP |
-Validation report
Summary document | emd_6334_validation.pdf.gz | 78.1 KB | Display | EMDB validaton report |
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Full document | emd_6334_full_validation.pdf.gz | 77.2 KB | Display | |
Data in XML | emd_6334_validation.xml.gz | 492 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6334 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-6334 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_6334.map.gz / Format: CCP4 / Size: 41.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Negative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.1 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Negative stain 3D reconstruction of the yeast 26S proteasome in A...
Entire | Name: Negative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein |
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Components |
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-Supramolecule #1000: Negative stain 3D reconstruction of the yeast 26S proteasome in A...
Supramolecule | Name: Negative stain 3D reconstruction of the yeast 26S proteasome in ATPgS in the presence of wild-type Ubp6 protein type: sample / ID: 1000 / Details: The sample was monodisperse. Oligomeric state: One to two 19S regulatory particles associates with the core particle to form a functional holoenzyme Number unique components: 1 |
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Molecular weight | Experimental: 1.5 MDa / Theoretical: 1.5 MDa |
-Macromolecule #1: 26S proteasome
Macromolecule | Name: 26S proteasome / type: protein_or_peptide / ID: 1 / Name.synonym: Proteasome Holoenzyme Details: Samples of 26S holoenzyme were incubated with wild-type Ubp6 protein in the presence of ATPgS, then diluted to ~25 nM for analysis by negative stain electron microscopy. Number of copies: 1 / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: YYS40 / synonym: yeast / Location in cell: cytoplasm |
Molecular weight | Experimental: 1.5 MDa / Theoretical: 1.5 MDa |
Sequence | GO: proteasome regulatory particle / InterPro: Proteasome, subunit alpha/beta |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.05 mg/mL |
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Buffer | pH: 7.6 Details: 60 mM HEPES, pH 7.6, 50 mM NaCl, 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 1 mM TCEP, 1 mM ATPgS |
Staining | Type: NEGATIVE Details: 4 uL of sample was applied to a freshly plasma-cleaned thin carbon surface pre-treated with 0.1% w/v poly-L-lysine hydrobromide. After removal of excess protein, negative staining was ...Details: 4 uL of sample was applied to a freshly plasma-cleaned thin carbon surface pre-treated with 0.1% w/v poly-L-lysine hydrobromide. After removal of excess protein, negative staining was performed using 2% w/v uranyl formate solution. |
Grid | Details: 400 mesh Cu-Rh Maxtaform grids were used following deposition of a thin continuous carbon film. |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI SPIRIT |
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Temperature | Min: 294 K / Max: 297 K / Average: 295 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected using a quadrupole stigmator at 52,000 times magnification. |
Date | Aug 26, 2014 |
Image recording | Category: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 2.5 µm / Number real images: 214 / Average electron dose: 20 e/Å2 Details: Automated imaging was performed using Leginon software. |
Tilt angle min | 0 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 52000 |
Sample stage | Specimen holder: Room temperature, side entry holder / Specimen holder model: SIDE ENTRY, EUCENTRIC |
Experimental equipment | Model: Tecnai Spirit / Image courtesy: FEI Company |
-Image processing
Details | The Appion software package was used for image processing leading to 3D reconstruction. Particles were selected from raw micrographs using the Difference of Gaussians (DoG)-based automated particle picker. The stack was subjected to five rounds of iterative 2D alignment and classification using multivariate statistical analysis (MSA) and multi-reference alignment (MRA). The selected particle stack was subjected to twenty-five iterations of 3D classification, requesting four classes using the Relion suite. Particles comprising well-resolved 3D class averages were used for further refinement by projection matching in Relion. |
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CTF correction | Details: Phase flipping of whole micrographs |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.2 Å / Resolution method: OTHER / Software - Name: Relion Details: Final 3D models were refined using 9000 particles selected by combining two of the 3D classes from Relion processing. Number images used: 9000 |
Final two d classification | Number classes: 4 |